More than 80 illnesses have been described that are associated with activation of auto-reactive lymphocytes and the production of autoantibodies directed against normal tissue or cellular components (autoantigens) [von Muhlen and Tan (1995) Semin Arthritis Rheum 24: 323-58; Mellors (2002) 2005]. Collectively referred to as autoimmune diseases, they are estimated to afflict 14.7-23.5 million people, up to 8% of the total U.S. population and constitute a major economic and health burden [Jacobson, Gange, Rose and Graham (1997) Clin Immunol Immunopathol 84: 223-43]. For unknown reasons, the number of people afflicted by autoimmune diseases is on the rise. An autoimmune diagnosis means a lifetime of illness and treatment, possible organ damage, debilitation and an increased chance of mortality. The chronic and often debilitating nature of autoimmune diseases results in poor patient health, increased medical costs, and decreased productivity. The root causes of the immune dysfunction underpinning autoimmune disease are still not well understood. Consequently, autoimmune diseases generally remain difficult to diagnose, due to the wide variability of clinical presentation, which typically involves a constellation of symptoms.
Autoimmune diseases are disorders in which an individual's immune system targets and destroys apparently normal tissue. Examples of autoimmune diseases include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), scleroderma (SCL), Sjogren's syndrome (SjS), polymyositis (PM), dermatomyositis (DM), mixed connective tissue disease (MCTD), pemphigus vulgaris (PV) and primary biliary cirrhosis (PBC). Autoantibodies are commonly directed against cellular proteins and nucleic acids. In certain diseases, such as PV, the target of autoantibodies is known and the autoantibody is thought to play a role in the pathogenesis of the disease. In other diseases, such as SLE, the targets of many different autoantibodies have been identified but the role of autoantibodies in the pathogenesis of SLE is as yet uncertain.
Detection of autoantibodies in the serum of patients assists in the diagnosis of autoimmune diseases. Rheumatoid factor (IgM antibodies directed against human IgG) is detected in the majority of patients with RA and supports that diagnosis in a given individual [Kelly, W. N., et al. 1985. Textbook of Rheumatology. 2nd ed. Saunders. pp. 667]. Antinuclear antibodies (ANA) are present in approximately 98% of individuals with active SLE. Although ANA are not specific for the diagnosis of SLE, the absence of these antibodies argues against the diagnosis of SLE in a given patient [Kelly et al., 1985 supra pp. 691].
Liver and biliary diseases collectively rank in the top ten causes of mortality in the U.S. Chronic liver diseases affect between 5 and 10 percent of Americans and cause 1 to 2 percent of deaths in the United States. Chronic liver disease and cirrhosis cost an estimated $1.6 billion per year [(2004)]. General causes of liver and biliary diseases include infectious agents, inherited defects, metabolic disturbances, alcohol, toxins and environmental toxicants. The most common liver diseases are chronic hepatitis C, alcohol liver disease, nonalcoholic fatty liver disease, chronic hepatitis B, autoimmune liver diseases and drug-induced liver diseases. Many of these conditions can be prevented or treated, but if not, they can lead to progressive liver injury, liver fibrosis and ultimately cirrhosis, portal hypertension, end-stage liver disease and, in some instances, liver cancer. Currently, the only therapy for end-stage liver disease is liver transplantation. More than 5,000 liver transplants are done in the U.S. each year. At least 17,000 persons are on a waiting list for liver transplantation and as many as 1,500 die yearly while waiting [(2004)]. Liver disease research presents many challenging needs. Autoimmune liver diseases include primary biliary cirrhosis (PBC), autoimmune hepatitis and primary sclerosing cholangitis. These chronic liver diseases can all lead to end-stage liver disease. Collectively, autoimmune liver diseases are responsible for 13% of adult liver transplants per year in the U.S. [(2004)].
PBC is a progressive cholestatic liver disease, with an estimated prevalence in the U.S. of approximately 40 adults per 100,000 population (incidence 2.7 per 100,000 U.S. population) [Kim, Lindor et al. (2000) Gastroenterology 119: 1631-6; Feld and Heathcote (2003) J Gastroenterol Hepatol 18: 1118-28; 2004)]. Women between the ages of 40 and 65 are predominantly affected by PBC, with a female to male ratio of 9:1 [Kaplan and Gershwin (2005) N Engl J Med 353: 1261-73], as is typical for autoimmune disease. PBC is characterized by the gradual progressive destruction of intrahepatic biliary ductules leading to hepatic fibrosis and liver failure (reviewed in [Kaplan (1996) N Engl J Med 335: 1570-80; Heathcote (2000) Hepatology 31: 1005-13; Kaplan (2002) Gastroenterology 123: 1392-4; Talwalkar and Lindor (2003) Lancet 362: 53-61]). PBC is a significant indication for liver transplantation, and PBC patients constitute 11% of all patients undergoing liver transplantation for cirrhosis [Milkiewicz (2008) Clin Liver Dis 12: 461-72; xi].
Treatment of PBC is accomplished with ursodeoxycholic acid (ursodiol), a natural bile acid that is not toxic to the liver, to replace the bile acids which are reduced by PBC. While the mechanisms are not fully understood, this treatment ultimately reduces intracellular build up of other liver-toxic bile acids (which was caused by bile duct destruction). Although ursodiol slows progression to cirrhosis, ursodiol treatment functions best when implemented early in the course of PBC, highlighting the importance of a rapid, reliable PBC diagnostic test. In fact, a study showed that ursodiol treatment at stages III and IV did not result in significant slowing of liver progression while patients treated early at histological stages I and II did show significant slowing of liver destruction with ursodiol treatment. This highlights the need for an early PBC diagnostic, to allow prompt medical treatment [Heathcote (2000) Hepatology 31: 1005-13; Poupon, Lindor, Pares, Chazouilleres, Poupon and Heathcote (2003) J Hepatol 39: 12-6].
Roughly half of PBC patients first present with an abnormal blood test which triggers the eventual PBC diagnosis. Generally, diagnostic testing is initially activated by abnormal liver function tests and signs of bile disease, followed by testing for serum anti-mitochondrial autoantibodies (AMA), for which an estimated 87-95% of PBC patients test positive [Heathcote (2000) Hepatology 31: 1005-13; Yang, Yu, Nakajima, Neuberg, Lindor and Bloch (2004) Clin Gastroenterol Hepatol 2: 1116-22; Kaplan and Gershwin (2005) N Engl J Med 353: 1261-73; Liu, Shi, Zhang, Zhang and Gao (2008) Liver Int 28: 233-9]. Bile duct imaging tests are used to rule out other causes of biliary tract disease, and liver biopsies confirm diagnosis and provide a gauge of disease stage (based upon the degree of fibrosis).
However, the other roughly half of PBC patients will present only with a variety of relatively non-specific physical symptoms, highlighting the difficulties facing the general practitioner or specialist responsible for diagnosis. The most common of such symptoms are pruritis, fatigue and musculoskeletal pain [Prince, Chetwynd, Newman, Metcalf and James (2002) Gastroenterology 123: 1044-51]. Furthermore, numerous autoimmune disorders may be found in association with PBC, including autoimmune hepatitis (AIH) [Czaja (2006) J Hepatol 44: 251-2], thyroid dysfunction, sicca symptoms, Raynaud's syndrome, systemic lupus erythematosus (SLE) and rheumatoid arthritis [Heathcote (2000) Hepatology 31: 1005-13; Gershwin, Selmi, Worman, Gold, Watnik, Utts, Lindor, Kaplan and Vierling (2005) Hepatology 42: 1194-202]. In one study, 19% of PBC patients were found to have features of another disease [Czaja (1998) Hepatology 28: 360-5], thereby clouding diagnosis. Of concern, the proper testing may not be ordered in many patients due to unrecognized etiology, especially when patients present with vague symptoms of pruritis or joint discomfort.
Autoantibodies have the potential to serve not only as diagnostic tools, but also as harbingers of the future development of PBC. In fact, anti-mitochondrial autoantibodies (AMA) have been shown to pre-date clinical manifestations and diagnosis of PBC [Metcalf, Mitchison, Palmer, Jones, Bassendine and James (1996) Lancet 348: 1399-402]. This demonstrates that it may be possible to diagnose PBC at an earlier stage using autoantibody biomarkers. The serological hallmark of PBC are AMA, which can be detected in 87-95% of patients [Kaplan (1996) N Engl J Med 335: 1570-80; Nishio, Keeffe and Gershwin (2002) Semin Liver Dis 22: 291-3021. The major autoantigens targeted by these AMA include the E2 subunits of the pyruvate dehydrogenase complex (PDC-E2), the branched/chain 2-oxo-acid dehydrogenase complex (BCOADC-E2) and the 2-oxo-glutarate dehydrogenase complex (OGDC-E2) [Fussey, Guest, James, Bassendine and Yeaman (1988) Proc Natl Acad Sci USA 85: 8654-8; Nishio, Keeffe et al. (2002) Semin Liver Dis 22: 291-302].
Anti-nuclear autoantibodies (ANA) are present in ˜50% of PBC patients. Autoantibodies recognizing proteins of the nuclear core complex and multiple nuclear dots (MND) are useful PBC markers in AMA-negative patients, with a prevalence of 13-44% [Manuel Lucena, Montes Cano, Luis Caro, Respaldiza, Alvarez, Sanchez-Roman, Nunez-Roldan and Wichmann (2007) Ann N Y Acad Sci 1109: 203-11]. Additionally, ANA can serve as prognostic indicators, with anti-centromere and/or anti-nuclear pore glycoprotein 210 (gp210) autoantibodies being associated with liver failure in PBC [Yang, Yu et al. (2004) Clin Gastroenterol Hepatol 2: 1116-22; Nakamura, Kondo et al. (2007) Hepatology 45: 118-27].
The nuclear body (NB, also known as nuclear domain 10, PML oncogenic domain, and Kr body) is a nuclear organelle whose function is unknown [Ascoli, C. A., and Maul, G. G., J. Cell. Biol. 112:785-795 (1991); Brasch, K., and Ochs, R. L., Exp. Cell Res. 202:211-223 (1992); Dyck, J. A. et al., Cell 76:333-343 (1994)]. Using immunohistochemical staining, NBs appear as 5 to 30 discrete, punctate, dot-like regions within the nucleus. The NB is distinct from other nuclear domains including those involved in DNA replication and mRNA processing. In addition, components of the NB do not co-localize with kinetochores or centromeres [Brasch, K., and Ochs, R. L., Exp. Cell Res. 202:211-223 (1992)]. The number of NBs in the cell, and the intensity of antibody staining of these structures, increase in response to stimuli including interferons (IFNs), heat shock and viral infection [Ascoli, C. A., and Maul, G. G., J. Cell. Biol. 112:785-795 (1991)].
The NB is a target of autoantibodies in the serum of patients with the autoimmune disease primary biliary cirrhosis (PBC). Approximately 40% of patients with PBC have antibodies directed against this structure [Evans, J., et al., Arthr. Rheum. 347:31-736 (1991); Szostecki, C. et al., Scand. J. Immunol. 36:555-564 (1992)]. Serum from patients with PBC was used to identify and characterize a 100-kDa component of the NB which was designated Sp100 (Speckled, 100 kDa) [Szostecki, C. et al., J. Immunol. 145:4338-4347 (1990)]. The fusion of Sp100 to the LexA DNA binding domain has been shown to activate gene transcription in Saccharomyces cerevisiae, and it has been suggested that Sp100 may participate in activation of transcription of specific regions in the genome [Xie, K. et al., Mol. Cell. Biol. 13:6170-6179 (1993)].
A second component of the NB, designated NDP52, was characterized using a murine monoclonal antibody that reacted with the NB [Korioth, F., et al., J. Cell. Biol. 130:1-13 (1995)]. A cDNA encoding NDP52 was identified and the predicted amino acid sequence contained coiled coil, leucine zipper and zinc finger motifs. One or more of these domains may be involved in interactions between NDP52 and other components of the NB [Korioth, F., et al., J. Cell. Biol. 130:1-13 (1995)].
A third component of the NB, PML, was identified by several investigators studying the t(15;17) translocation associated with human acute promyelocytic leukemia (APL) [de The, H. et al., Nature (London) 347:558-561 (1990); Borrow, J. et al., Science 249:1577-1580 (1990); Longo, L. et al., J. Exp. Med. 172:1571-1575 (1990); Kakizuka, A. et al., Cell 66:663-674 (1991)]. In this translocation, the amino terminal portion of PML is fused to retinoic acid receptor alpha. PML was found to co-localize with Sp100 in the NB [Weis, K. et al., Cell 76:345-356 (1994); Koken, M. H. M. et al., EMBO 13:1073-1083 (1994)]. Expression of the PML-alpha fusion protein in APL cells appears to disrupt the NB; in these cells, the NB antigens are detected in numerous smaller regions in the nucleus described as “microspeckles.” Treatment of APL cells with retinoic acid (RA) results in differentiation of myeloid precursor cells and reformation of NBs [Dyck, J. A. et al., Cell 76:333-343 (1994); Weis, K. et al., Cell 76:345-356 (1994); Koken, M. H. M. et al., EMBO 13:1073-1083 (1994)]. In patients with APL, treatment with RA results in differentiation of leukemic cells and temporary disease remission [Warrell, R. P. et al., N. Eng. J. Med. 329:177-189 (1993)].
It is important to note however, that ANA are also found in a variety of other prevalent autoimmune disorders and a wide range of cancers [Bei, Masuelli, Palumbo, Modesti and Modesti (2008) Cancer Lett].
Indirect immunofluorescence (IIF) and solid-phase immunoassay are the two formats used to establish the presence or absence of autoantibodies in patients. Both methods have their pros and cons as discussed below:
For the past several decades, indirect immunofluorescence (IIF) has been the method of choice by physicians for the detection of autoantibodies present in the serum of autoimmune patients. Importantly, it remains the gold standard for AMA and ANA testing, including for PBC. Typically, patient serum is serial diluted in two-fold increments and allowed to bind to a cell substrate on a microscope slide (e.g. HEp-2 liver cells), which is then fluorescently stained to detect bound autoantibodies and examined under the microscope by a trained technician to identify the cellular/tissue staining patterns. IIF does have the advantage that as a cell/tissue based substrate, it can in theory “universally” cover all cellular autoantigens (pending their expression and preservation in the substrate). This, in part, is evidenced by the high diagnostic sensitivity of the IIF test, e.g. 93% (ANA) for systemic lupus erythematosus (SLE) [Solomon, Kavanaugh and Schur (2002) Arthritis Rheum 47: 434-44] and 90% (AMA) for PBC [Tanaka, Miyakawa, Luketic, Kaplan, Storch and Gershwin (2002) Cell Mol Biol (Noisy-le-grand) 48: 295-9].
Although IIF based AMA is a sensitive marker for PBC, the tradeoff may be specificity. Asymptomatic patients have been deemed AMA positive, and while a large portion only develop symptoms years later, some never develop symptoms at all [Metcalf, Mitchison et al. (1996) Lancet 348: 1399-402]. Moreover, one study found that 34% of AIH patients tested positive for AMA [Nezu, Tanaka, Yasui, Imamura, Nakajima, Ishida and Takahashi (2006) J Gastroenterol Hepatol 21: 1448-54].
Furthermore, the IIF assay is problematic overall when used as a routine diagnostic screening tool, as it is difficult to standardize owing to variations in the substrate and fixation process variations in the microscopy apparatus and due to the highly subjective interpretation of results [Jaskowski, Schroder, Martins, Mouritsen, Litwin and Hill (1996) Am J Clin Pathol 105: 468-73]. The consensus statement in 2004 from the Committee for Autoimmune Serology of the International Autoimmune Hepatitis Group (IAIHG) recommended that IIF be performed on three different organs from rodents [Vergani, Alvarez, Bianchi, Cancado, Mackay, Maims, Nishioka and Penner (2004) J Hepatol 41: 677-83]. Both AMA and anti-liver kidney microsomal-1 (LKM1) antibodies stain the renal tubules of the kidney, with differences only apparent to the trained eye, and this confusion can lead to a diagnosis of autoimmune hepatitis (AIR) instead of PBC [Bogdanos, Invernizzi, Mackay and Vergani (2008) World J Gastroenterol 14: 3374-87]. Moreover, some autoantigens are lost (unrecognizable) by diffusion or denaturation during the fixation process of IIF. Another confounding factor is that multiple autoimmune diseases can often occur together in the same patient, and the overlapping IIF patterns can lead to confusion in the correct diagnosis of each [Assassi, Fritzler et al. (2009) J Rheumatol; Norman, Bialek, Encabo, Butkiewicz, Wiechowska-Kozlowska, Brzosko, Shums and Milkiewicz (2009) Dig Liver Dis 41: 762-4]. Finally, IIF is slow, laborious and not amenable to high-throughput automation [Ulvestad, Kanestrom, Madland, Thomassen, Haga and Vollset (2000) Scand J Immunol 52: 309-15].
Although IIF remains the gold standard in AMA testing, solid-phase immunoassays, such as ELISA (Enzyme Linked Immunosorbent Assay), are gaining popularity, especially in high-throughput laboratories [Fritzler and Fritzler (2006) Curr Med Chem 13: 2503-12]. These methods have the advantage of high throughput automation, high analytical sensitivity, purely objective scoring, reliability, and the ability to test for specific autoantigen species, including in a multiplexed fashion [Fritzler and Fritzler (2006) Curr Med Chem 13: 2503-12]. With a resolution at the individual antigen level, these methods have the potential for greater disease specificity, if the correct marker panel is chosen. The drawback, however, is that a sufficient number of autoantigens needs to be both discovered and clinically validated to match the diagnostic sensitivity of the cellular substrate based IIF assay.
In one example of a commercial solid-phase immunoassay for PBC, INOVA Diagnostics Inc. (San Diego, Calif.) markets the MIT3 assay, an FDA-approved ELISA-based immunoassay for PBC based on the detection of AMAs. The MIT3 is utilizes a recombinant protein containing the immunodominant epitopes of all three E2 subunits of the pyruvate dehydrogenase complex [Moteki, Leung, Coppel, Dickson, Kaplan, Munoz and Gershwin (1996) Hepatology 24: 97-103]. The overall goal of these tests is to mimic the cellular IIF-based AMA test for PBC, but with all the aforementioned benefits of solid-phase immunoassays of individual antigens. Still, this test is only meant to be diagnostic aid, together with clinicopathological findings for PBC. In one study, the AMA-based MIT3 ELISA assay had a reported a diagnostic sensitivity of 81.6%, however, it is important to note that serum samples with AMA-negative PBC disease were excluded [Gabeta, Norman, Liaskos, Papamichalis, Zografos, Garagounis, Rigopoulou and Dalekos (2007) J Clin Immunol 27: 378-87]. In another study, it was shown that the MIT3 assay, for instance, lacks all the necessary mitochondrial autoantigens for maximum diagnostic sensitivity of PBC [Dahnrich, Pares et al. (2009) Clin Chem 55: 978-85].
This highlights the need for the discovery and validation of additional autoantigen biomarkers to be used in solid-phase immunoassays for the optimal diagnosis of autoimmune diseases such as PBC. The most effective methods for the discovery of autoantigens are proteomics based. Proteomics can be defined as the global (e.g. parallel or simultaneous) analysis of the entire expressed protein compliment of the genome [Wasinger, Cordwell et al. (1995) Electrophoresis 16: 1090-4]. Proteomics methods allow for the discovery of novel autoantigens in an unbiased fashion. Common proteomics methods for discovery of novel autoantigens include SEREX (serological identification of antigens by recombinant expression cloning) [Krebs, Kurrer, Sahin, Tureci and Ludewig (2003) Autoimmun Rev 2: 339-45] and human proteome microarrays (“chips”, commonly the dimensions of standard microscope slides, containing thousands of purified recombinant human proteins printed to their surface in an ordered array of microscopic spots, e.g. spots of 100 micron in diameter) [Robinson, DiGennaro et al. (2002) Nat Med 8: 295-301; Robinson, Steinman and Utz (2002) Arthritis Rheum 46: 885-93].